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Slime production and adherence by coagulase-negative staphylococci
327
Letters to the Editor effects
of this regimen
the risk of selecting
in neutropenic
less susceptible
G. E. Bignardi M. A. Savage R. Coker* S. G. Davis?
patients strains
should
be weighed
against
such as C. izrusei.
Department of Medical Microbiology *Department of Genito- Urinary Medicine and 7 Department of Haematology St Mary’s Hospital London W2 1NY References
1. Jakab K, Kelemen E, Prinz G, T&ok I. Amphotericin-resistant invasive hepatosplenic candidiasis controlled by fluconazole. Lancet 1990; 335: 473474. 2. Conti DJ, Tolkoff-Rubin NE, Baker GP, Doran M, Cosimi AB, Delmonico F et al. Successful treatment of invasive fungal infection with fluconazole in organ transplant recipients. Transplantation 1989; 48: 692-695. 3. Marriott MS, Richardson K. The discovery and mode of action of fluconazole. In: Development and Edaluation of Fromling RA, Ed. Recent Trends in the Discovery, Antifungal Agents. J R Prous Science Publishers 1987; 81-92. 4. Merz WG, Karp JE, Schron D, Sara1 R. Increased incidence of fungemia caused by Candida krusei. 7 Clin Microbial 1986: 24: 581-584. 5. Fisher MA, Sh;eh-Hui S, Haddad J, Tarry WF. Comparison of in vivo activity of fluconazole with that of amphotericin B against Candida tropicalis, Candida glabrata and Candida krusei. Antimicrob Agents Chemother 1989; 33: 1443-6. 6. Just G, Steinheimer D, Schnellbach B, Bottinger C, Helm EB, Stille W. Change of causative organisms under antifungal treatment in immunosuppressed patients with HIV-infections. Mycoses 1989; 32 (Suppl 2): 47-51.
Sir,
Slime
production
and adherence by coagulase-negative staphylococci
We read with interest the study of phenotypic characteristics in S. epidermidis by Souto et al.’ and in particular of their attempt to examine the phenomena of adherence and slime production. The fault of most in-vitro assays of slime production by coagulase-negative staphylococci (CNS) is the reliance on initial bacterial adherence for a positive result. As such, strains that may be able to produce slime and yet not adhere to the surface under investigation (see below) are ignored. Souto et al.’ examined slime production by planktonic cells using a lectin (concanavalin A) based assay’ and the same phenomenon by adherent bacteria in the tube test.3 We have similarly found a lack of correlation between these two assays. We also employed three other lectins (Machra pomifera, Pisum sat&urn and poly-L-lysine) but failed to correlate these results with those of S. epidermidis adherence as measured by the tube,3 microtitre tray,4 ATP
Letters
328
to the Editor
bioluminescence or “C-glucose assays.’ Interestingly, our slide agglutination results remained unaltered after prior washing (three times) of bacteria in phosphate-buffered saline. This procedure should be sufficient to remove the loosely bound, water soluble extracellular slime from the bacterial cells. Our observations imply that the lectins may be binding to sugars present in the cell wall as opposed to slime material. Souto et al.’ refer to earlier work reporting a correlation between lectin reactivity and adherence for a single S. epidermidis strain.2 In fact, that study noted a marked media dependent variability in the results of these
Figure air.
1. Slime production
by RP62A
(A) and C362 (B) cultured
in tryptone
soya broth
in
Letters
to the Editor
329
assays. Indeed, despite a S-fold difference in the number of attached bacteria in one medium compared to another, the degree of agglutination with concanavalin A was the same. The adherence of CNS is not only media dependent, but has also been shown to be polymerand atmosphere-specific in some cases.6-8 Hence, the use of culture vessels made of glass or polystyrene (materials that are not employed in the manufacture of medical devices) in assays of CNS adherence is not to be recommended when attempting a correlation with virulence. Similarly, it is desirable that atmospheric culture conditions reflect the in viva situation. 6,7,9 The failure to take such factors into consideration may explain the discrepant results of studies attempting to correlate adherence (or slime production) by CNS with virulence in vitro.“,”
Figure dialysis
2. Adherence to polystyrene fluid in air with 5% CO,.
of RP62A
(A)
and C362
(B) cultured
in peritoneal
330
Letters
to the Editor
We have studied the adherence (to polystyrene segments suspended in media) and slime production by planktonic cells of two S. epidermidis strains by scanning and transmission electron microscopy (SEM and TEM), respectively. The photographs shown in Figures l-3 illustrate the problems discussed above. Strain RP62A (ATCC 35984) is tube adherence test-positive when cultured in tryptone soya broth (TSB) in air,3 while C362 is negative; these observations correlated with appearances by SEM.
Figure 3. Slime production in air with 5% CO,.
by RP62A
(A) and C362 (B) cultured
in peritoneal
dialysis
fluid
Letters
to the Editor
331
However, both strains clearly produced slime in TSB as demonstrated by TEM (Figures 1A and 1B). When these strains were cultured instead in pooled, used, peritoneal dialysis fluid, collected from patients undergoing continuous ambulatory peritoneal dialysis, in air with 5% CO, (to simulate the carbon dioxide tension and pH found in the peritoneal cavity)’ very different results were obtained. C362 adhered better than RP62A (Figures 2A and 2B) and yet slime production, at a much reduced level when compared with culture in TSB, was only evident in the latter strain (Figures 3A and 3B). to adhere first to catheter et a1.12 noted that CNS appeared Peters segments and then produce slime. They did not, however, examine planktonic cells for slime production. Nevertheless, it is apparent that the production of slime and adherence to polymer surfaces are two separate processes. The mechanisms involved in CNS adherence are clearly complex, and as yet have not been fully identified. Slime production may play a part in consolidating bacterial accumulation once adherence has occurred; it is certainly not the whole story.
M. M. M. P. R.
Departments of Experimental and Clinical Microbiology, Molecular Biology and Biotechnology, and Pathology University of Shefield Medical School Beech Hill Road Shefield SlO 2RX
H. Wilcox Hussain K. Faulkner J. White C. Spencer
References CM, Criado MT. Failure of phenotypic characteristics to 1. Souto MJ, Ferreiros distinnuish between carrier and invasive isolates of Staphylococcus epidevmidis. _.r Hasp_ _ Infect-1991; 17: 107-115. A, Uhlenbruck G, Peters G et al. Investigation on extracellular slime 2. Ludwicka substance produced by Staphylococcus epidermidis. Zentralbl Bakt Hyg A 1984; 2.58: 2566267. GD. Simnson WA. Bisno AL. Beachev EH. Adherence of slime-producing 3. Christenson strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982; 371318-326. GD, Simpson WA, Younger JJ et al. Adherence of coagulase-negative 4. Christenson staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbial 1985; 22: 996-1006. assay to measure the 5. Hussain M, Collins C, Hastings JGM, White PJ. A radiochemical adherence of coagulase-negative staphylococci to glass surfaces and its use to quantitate the effects of various antibacterial compounds on attachment. J Med Microbial 1991; in press. SP, Davies MC, Evans JA et al. Influence of carbon dioxide on the surface 6. Denyer characteristics and adherence notential of coagulase-negative staphvlococci. 7 Clin Microbial 1990; 28: 181331817. P. Effects of carbon dioxide 7. Wilcox MH. Denver SP. Finch RG. Smith DGE. Williams and sub-lethal levels of antibiotics on adherence of coagulase-negative staphylococci to polystyrene and silicone rubber. J Antimicrob Chemoth& 1991; 27: 577-587.. onto biomaterials. in: Pulverer 8. Hogt AH, Dankert J, Feijen J. Adhesion of staphylococci G, Quie PG, Peters G, Eds. Pathogenicity and Clinical Significance of Coagulase-negative Staphylococci. Zentralbl Bakt (Suppl. 16). Stuttgart: Gustav Fischer Verlag 1987; 113-131. I
i
332
Letters
to the Editor
9. Wilcox MH, Smith DGE, Evans JA, Denyer SP, Finch RG, Williams P. Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate. J Clin Microbial 1990; 28: 2183-2186. 10. Kristinsson KG, Spencer RC, Brown CB. Clinical importance of production of a slime by coagulase-negative staphylococci in chronic ambulatory peritoneal dialysis. r Clin Path 1986; 39: 117-118. 11. Needham CA, Stempsey W. Incidence, adherence and antibiotic resistance of coagulase-negative Staphylococus species causing human disease. Diagn Microbial Infect Dis 1984; 2: 293-299. 12. Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. J Infect Dis 1982; 146: 479-482.
Sir,
Hospital-acquired
meningococcaemia
In October 1990 a mother developed meningococcaemia 4 days after nursing her child with meningococcaemia and meningitis. The index patient was a 3&year-old girl who was admitted to the Intensive Care Unit of a children’s hospital. During her stay in the hospital she had high grade fever, vomited several times, and had a petechial rash. Blood and CSF culture grew group A Neisseria meningitidis. A Gram smear from the petechial rash had shown Gram-negative diplococci. The isolate was resistant to sulphonamides. Four days after the index patient was admitted to the hospital the 27-year-old mother of the child developed headache, fever and malaise. She had been nursing her child very closely during this time. Two days after onset of her symptoms she presented for examination, and was noted to have scattered petechial lesions on her arms and legs. A lumbar puncture showed normal CSF. Over the next several days she developed a more severe headache, more petechiae and was admitted to the hospital with a presumptive diagnosis of meningococcaemia..A second blood culture grew group A N. meningitidis that was not susceptible to sulphonamides. The patient recalled that her child was restless and that she had nursed the child for some days; and had received sulphadiazine as prophylaxis. There was no known contact with other persons with meningococcal disease. We report this case as an acquired infection with N. meningitidis from a patient with meningococcaemia and meningitis. To our knowledge, this is the first report of its kind from India.
V. L. Malhotra
Department of Microbiology, Lady Harding Medical College, New Delhi 110001, India
Letters to the Editor effects
of this regimen
the risk of selecting
in neutropenic
less susceptible
G. E. Bignardi M. A. Savage R. Coker* S. G. Davis?
patients strains
should
be weighed
against
such as C. izrusei.
Department of Medical Microbiology *Department of Genito- Urinary Medicine and 7 Department of Haematology St Mary’s Hospital London W2 1NY References
1. Jakab K, Kelemen E, Prinz G, T&ok I. Amphotericin-resistant invasive hepatosplenic candidiasis controlled by fluconazole. Lancet 1990; 335: 473474. 2. Conti DJ, Tolkoff-Rubin NE, Baker GP, Doran M, Cosimi AB, Delmonico F et al. Successful treatment of invasive fungal infection with fluconazole in organ transplant recipients. Transplantation 1989; 48: 692-695. 3. Marriott MS, Richardson K. The discovery and mode of action of fluconazole. In: Development and Edaluation of Fromling RA, Ed. Recent Trends in the Discovery, Antifungal Agents. J R Prous Science Publishers 1987; 81-92. 4. Merz WG, Karp JE, Schron D, Sara1 R. Increased incidence of fungemia caused by Candida krusei. 7 Clin Microbial 1986: 24: 581-584. 5. Fisher MA, Sh;eh-Hui S, Haddad J, Tarry WF. Comparison of in vivo activity of fluconazole with that of amphotericin B against Candida tropicalis, Candida glabrata and Candida krusei. Antimicrob Agents Chemother 1989; 33: 1443-6. 6. Just G, Steinheimer D, Schnellbach B, Bottinger C, Helm EB, Stille W. Change of causative organisms under antifungal treatment in immunosuppressed patients with HIV-infections. Mycoses 1989; 32 (Suppl 2): 47-51.
Sir,
Slime
production
and adherence by coagulase-negative staphylococci
We read with interest the study of phenotypic characteristics in S. epidermidis by Souto et al.’ and in particular of their attempt to examine the phenomena of adherence and slime production. The fault of most in-vitro assays of slime production by coagulase-negative staphylococci (CNS) is the reliance on initial bacterial adherence for a positive result. As such, strains that may be able to produce slime and yet not adhere to the surface under investigation (see below) are ignored. Souto et al.’ examined slime production by planktonic cells using a lectin (concanavalin A) based assay’ and the same phenomenon by adherent bacteria in the tube test.3 We have similarly found a lack of correlation between these two assays. We also employed three other lectins (Machra pomifera, Pisum sat&urn and poly-L-lysine) but failed to correlate these results with those of S. epidermidis adherence as measured by the tube,3 microtitre tray,4 ATP
Letters
328
to the Editor
bioluminescence or “C-glucose assays.’ Interestingly, our slide agglutination results remained unaltered after prior washing (three times) of bacteria in phosphate-buffered saline. This procedure should be sufficient to remove the loosely bound, water soluble extracellular slime from the bacterial cells. Our observations imply that the lectins may be binding to sugars present in the cell wall as opposed to slime material. Souto et al.’ refer to earlier work reporting a correlation between lectin reactivity and adherence for a single S. epidermidis strain.2 In fact, that study noted a marked media dependent variability in the results of these
Figure air.
1. Slime production
by RP62A
(A) and C362 (B) cultured
in tryptone
soya broth
in
Letters
to the Editor
329
assays. Indeed, despite a S-fold difference in the number of attached bacteria in one medium compared to another, the degree of agglutination with concanavalin A was the same. The adherence of CNS is not only media dependent, but has also been shown to be polymerand atmosphere-specific in some cases.6-8 Hence, the use of culture vessels made of glass or polystyrene (materials that are not employed in the manufacture of medical devices) in assays of CNS adherence is not to be recommended when attempting a correlation with virulence. Similarly, it is desirable that atmospheric culture conditions reflect the in viva situation. 6,7,9 The failure to take such factors into consideration may explain the discrepant results of studies attempting to correlate adherence (or slime production) by CNS with virulence in vitro.“,”
Figure dialysis
2. Adherence to polystyrene fluid in air with 5% CO,.
of RP62A
(A)
and C362
(B) cultured
in peritoneal
330
Letters
to the Editor
We have studied the adherence (to polystyrene segments suspended in media) and slime production by planktonic cells of two S. epidermidis strains by scanning and transmission electron microscopy (SEM and TEM), respectively. The photographs shown in Figures l-3 illustrate the problems discussed above. Strain RP62A (ATCC 35984) is tube adherence test-positive when cultured in tryptone soya broth (TSB) in air,3 while C362 is negative; these observations correlated with appearances by SEM.
Figure 3. Slime production in air with 5% CO,.
by RP62A
(A) and C362 (B) cultured
in peritoneal
dialysis
fluid
Letters
to the Editor
331
However, both strains clearly produced slime in TSB as demonstrated by TEM (Figures 1A and 1B). When these strains were cultured instead in pooled, used, peritoneal dialysis fluid, collected from patients undergoing continuous ambulatory peritoneal dialysis, in air with 5% CO, (to simulate the carbon dioxide tension and pH found in the peritoneal cavity)’ very different results were obtained. C362 adhered better than RP62A (Figures 2A and 2B) and yet slime production, at a much reduced level when compared with culture in TSB, was only evident in the latter strain (Figures 3A and 3B). to adhere first to catheter et a1.12 noted that CNS appeared Peters segments and then produce slime. They did not, however, examine planktonic cells for slime production. Nevertheless, it is apparent that the production of slime and adherence to polymer surfaces are two separate processes. The mechanisms involved in CNS adherence are clearly complex, and as yet have not been fully identified. Slime production may play a part in consolidating bacterial accumulation once adherence has occurred; it is certainly not the whole story.
M. M. M. P. R.
Departments of Experimental and Clinical Microbiology, Molecular Biology and Biotechnology, and Pathology University of Shefield Medical School Beech Hill Road Shefield SlO 2RX
H. Wilcox Hussain K. Faulkner J. White C. Spencer
References CM, Criado MT. Failure of phenotypic characteristics to 1. Souto MJ, Ferreiros distinnuish between carrier and invasive isolates of Staphylococcus epidevmidis. _.r Hasp_ _ Infect-1991; 17: 107-115. A, Uhlenbruck G, Peters G et al. Investigation on extracellular slime 2. Ludwicka substance produced by Staphylococcus epidermidis. Zentralbl Bakt Hyg A 1984; 2.58: 2566267. GD. Simnson WA. Bisno AL. Beachev EH. Adherence of slime-producing 3. Christenson strains of Staphylococcus epidermidis to smooth surfaces. Infect Immun 1982; 371318-326. GD, Simpson WA, Younger JJ et al. Adherence of coagulase-negative 4. Christenson staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbial 1985; 22: 996-1006. assay to measure the 5. Hussain M, Collins C, Hastings JGM, White PJ. A radiochemical adherence of coagulase-negative staphylococci to glass surfaces and its use to quantitate the effects of various antibacterial compounds on attachment. J Med Microbial 1991; in press. SP, Davies MC, Evans JA et al. Influence of carbon dioxide on the surface 6. Denyer characteristics and adherence notential of coagulase-negative staphvlococci. 7 Clin Microbial 1990; 28: 181331817. P. Effects of carbon dioxide 7. Wilcox MH. Denver SP. Finch RG. Smith DGE. Williams and sub-lethal levels of antibiotics on adherence of coagulase-negative staphylococci to polystyrene and silicone rubber. J Antimicrob Chemoth& 1991; 27: 577-587.. onto biomaterials. in: Pulverer 8. Hogt AH, Dankert J, Feijen J. Adhesion of staphylococci G, Quie PG, Peters G, Eds. Pathogenicity and Clinical Significance of Coagulase-negative Staphylococci. Zentralbl Bakt (Suppl. 16). Stuttgart: Gustav Fischer Verlag 1987; 113-131. I
i
332
Letters
to the Editor
9. Wilcox MH, Smith DGE, Evans JA, Denyer SP, Finch RG, Williams P. Influence of carbon dioxide on growth and antibiotic susceptibility of coagulase-negative staphylococci cultured in human peritoneal dialysate. J Clin Microbial 1990; 28: 2183-2186. 10. Kristinsson KG, Spencer RC, Brown CB. Clinical importance of production of a slime by coagulase-negative staphylococci in chronic ambulatory peritoneal dialysis. r Clin Path 1986; 39: 117-118. 11. Needham CA, Stempsey W. Incidence, adherence and antibiotic resistance of coagulase-negative Staphylococus species causing human disease. Diagn Microbial Infect Dis 1984; 2: 293-299. 12. Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. J Infect Dis 1982; 146: 479-482.
Sir,
Hospital-acquired
meningococcaemia
In October 1990 a mother developed meningococcaemia 4 days after nursing her child with meningococcaemia and meningitis. The index patient was a 3&year-old girl who was admitted to the Intensive Care Unit of a children’s hospital. During her stay in the hospital she had high grade fever, vomited several times, and had a petechial rash. Blood and CSF culture grew group A Neisseria meningitidis. A Gram smear from the petechial rash had shown Gram-negative diplococci. The isolate was resistant to sulphonamides. Four days after the index patient was admitted to the hospital the 27-year-old mother of the child developed headache, fever and malaise. She had been nursing her child very closely during this time. Two days after onset of her symptoms she presented for examination, and was noted to have scattered petechial lesions on her arms and legs. A lumbar puncture showed normal CSF. Over the next several days she developed a more severe headache, more petechiae and was admitted to the hospital with a presumptive diagnosis of meningococcaemia..A second blood culture grew group A N. meningitidis that was not susceptible to sulphonamides. The patient recalled that her child was restless and that she had nursed the child for some days; and had received sulphadiazine as prophylaxis. There was no known contact with other persons with meningococcal disease. We report this case as an acquired infection with N. meningitidis from a patient with meningococcaemia and meningitis. To our knowledge, this is the first report of its kind from India.
V. L. Malhotra
Department of Microbiology, Lady Harding Medical College, New Delhi 110001, India